EP0569527B1 - Mutations hereditaires et somatiques du gene apc dans les cancers recto-coliques chez l'homme - Google Patents

Mutations hereditaires et somatiques du gene apc dans les cancers recto-coliques chez l'homme Download PDF

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EP0569527B1
EP0569527B1 EP92906080A EP92906080A EP0569527B1 EP 0569527 B1 EP0569527 B1 EP 0569527B1 EP 92906080 A EP92906080 A EP 92906080A EP 92906080 A EP92906080 A EP 92906080A EP 0569527 B1 EP0569527 B1 EP 0569527B1
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apc
wild
gene
type
apc gene
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EP0569527A1 (fr
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Kenneth W. Kinzler
Bert Vogelstein
Rakesh Anand
Philip John Hedge
Alexander Fred Markham
Hans Albertsen
Mary L. Carlson
Joanna L. Groden
Geoff Joslyn
Andrew Thliveris
Raymond L. White
Yusuke Nakamura
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Johns Hopkins University En Syngenta Ltd E
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Zeneca Ltd
Japanese Foundation for Cancer Research
Johns Hopkins University
University of Utah
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the invention relates to the area of cancer diagnostics and therapeutics. More particularly, the invention relates to detection of the germline and somatic alterations of wild-type APC genes. In addition, it relates to therapeutic intervention to restore the function of APC gene product.
  • FAP is an autosomal-dominant, inherited disease in which affected individuals develop hundreds to thousands of adenomatous polyps, some of which progress to malignancy.
  • GS is a variant of FAP in which desmoid tumors, osteomas and other soft tissue tumors occur together with multiple adenomas of the colon and rectum.
  • a less severe form of polyposis has been identified in which only a few (2-40) polyps develop. This condition also is familial and is linked to the same chromosomal markers as FAP and GS (Leppert et al., New England Journal of Medicine, Vol. 322, pp.
  • this chromosomal region is often deleted from the adenomas (Vogelstein et al., N. Engl. J. Med., Vol. 319, p. 525 (1988)) and carcinomas (Vogelstein et al., N. Engl. J. Med., Vol. 319, p. 525 (1988); Solomon et al., Nature, Vol. 328, p. 616 (1987); Sasaki et al., Cancer Research, Vol. 49, p. 4402 (1989); Delattre et al., Lancet, Vol. 2, p. 353 (1989); and Ashton-Rickardt et al., Oncogene, Vol.
  • the MCC gene has been identified on 5q21 as a candidate suppressor gene, it does not appear to be altered in FAP or GS patients. Thus there is a need in the art for investigations of this chromosomal region to identify genes and to determine if any of such genes are associated with FAP and/or GS and the process of tumorigenesis.
  • a method of diagnosing or prognosing a neoplastic tissue of a human comprising: detecting somatic alteration of wild-type APC genes or their expression products in a sporadic colorectal cancer tissue, said alteration indicating neoplasia of the tissue.
  • an in vitro method of detecting genetic predisposition to cancer in a human including familial adenomatous polyposis (FAP) and Gardner's Syndrome (GS), comprising: using an isolated human sample selected from the group consisting of blood and fetal tissue; detecting alteration of wild-type APC gene coding sequences or their expression products from the sample, said alteration indicating genetic predisposition to cancer.
  • FAP familial adenomatous polyposis
  • GS Gardner's Syndrome
  • a wild-type APC gene for use in a method for supplying wild-type APC gene function to a cell which has lost said gene function by virtue of a mutation in the APC gene, said method comprising: introducing a wild-type APC gene into a cell which has lost said gene function such that said wild-type gene is expressed in the cell.
  • APC protein can also be applied to cells or administered to animals to remediate for mutant APC genes. Synthetic peptides or drugs can also be used to mimic APC function in cells which have altered APC expression.
  • a pair of single stranded primers is provided for determination of the nucleotide sequence of the APC gene by polymerase chain reaction.
  • the sequence of said pair of single stranded DNA primers is derived from chromosome 5q band 21, said pair of primers allowing synthesis of APC gene coding sequences.
  • nucleic acid probe which is complementary to human wild-type APC gene coding sequences and which can form mismatches with mutant APC genes, thereby allowing their detection by enzymatic or chemical cleavage or by shifts in electrophoretic mobility.
  • a method for detecting the presence of a neoplastic tissue in a human comprises the use of an isolated body sample from a human; detecting in said sample alteration of a wild-type APC gene sequence or wild-type APC expression product, said alteration indicating the presence of a neoplastic tissue in the human.
  • a cDNA molecule which comprises the coding sequence of the APC gene.
  • a preparation of the human APC protein is provided which is substantially free of other human proteins.
  • the amino acid sequence of the protein is shown in Figure 3 or 7.
  • an in vitro method for screening for genetic predisposition to cancer, including familial adenomatous polyposis (FAP) and Gardner's Syndrome (GS), in a human.
  • the method comprises: detecting among kindred persons the presence of a DNA polymorphism which is linked to a mutant APC allele in an individual having a genetic predisposition to cancer, said kindred being genetically related to the individual, the presence of said polymorphism suggesting a predisposition to cancer.
  • a method of testing therapeutic agents for the ability to suppress a neoplastically transformed phenotype comprises: applying a test substance to a cultured epithelial cell which carries a mutation in an APC allele; and determining whether said test substance suppresses the neoplastically transformed phenotype of the cell.
  • a method of testing therapeutic agents for the ability to suppress a neoplastically transformed phenotype comprises: administering a test substance to an animal to the exclusion of humans which carries a mutant APC allele; and determining whether said test substance prevents or suppresses the growth of tumors.
  • transgenic animals are provided.
  • the animals carry a mutant APC allele from a second animal species or have been genetically engineered to contain an insertion mutation which disrupts an APC allele.
  • the present invention provides the art with the information that the APC gene, a heretofore unknown gene is, in fact, a target of mutational alterations on chromosome 5q21 and that these alterations are associated with the process of tumorigenesis.
  • This information allows highly specific assays to be performed to assess the neoplastic status of a particular tissue or the predisposition to cancer of an individual.
  • This invention has applicability to Familial Adenomatous Polyposis, sporadic colorectal cancers, Gardner's Syndrome, as well as the less severe familial polyposis discusses above.
  • Figure 1A shows an overview of yeast artificial chromosome (YAC) contigs. Genetic distances between selected RFLP markers from within the contigs are shown in centiMorgans.
  • YAC yeast artificial chromosome
  • Figure 1B shows a detailed map of the three central contigs. The position of the six identified genes from within the FAP region is shown; the 5' and 3' ends of the transcripts from these genes have in general not yet been isolated, as indicated by the string of dots surrounding the bars denoting the genes' positions. Selected restriction endonuclease recognition sites are indicated. B, BssH2; S, SstII; M, MluI; N, NruI.
  • Figure 2 shows the sequence of TB1 and TB2 genes.
  • the cDNA sequence of the TB1 gene was determined from the analysis of 11 cDNA clones derived from normal colon and liver, as described in the text. A total of 2314 bp were contained within the overlapping cDNA clones, defining an ORF of 424 amino acids beginning at nucleotide 1. Only the predicted amino acids from the ORF are shown. The carboxy-terminal end of the ORF has apparently been identified, but the 5' end of the TB1 transcript has not yet been precisely determined.
  • the cDNA sequence of the TB2 gene was determined from the YS-39 clone derived as described in the text. This clone consisted of 2300 bp and defined an ORF of 185 amino acids beginning at nucleotide 1. Only the predicted amino acids are shown. The carboxy terminal end of the ORF has apparently been identified, but the 5' end of the TB2 transcript has not been precisely determined.
  • Figure 3 shows the sequence of the APC gene product.
  • the cDNA sequence was determined through the analysis of 87 cDNA clones derived from normal colon, liver, and brain. A total of 8973 bp were contained within overlapping cDNA clones, defining an ORF of 2842 amino acids. In frame stop codons surrounded this ORF, as described in the text, suggesting that the entire APC gene product was represented in the ORF illustrated. Only the predicted amino acids are shown.
  • Figure 4 shows the local similarity between human APC and ral2 of yeast. Local similarity among the APC and MCC genes and the m3 muscarinic acetylcholine receptor is shown. The region of the mAChR shown corresponds to that responsible for coupling the receptor to G proteins. The connecting lines indicate identities; dots indicate related amino acids residues.
  • Figure 5 shows the genomic map of the 1200 kb NotI fragment at the FAP locus.
  • the NotI fragment is shown as a bold line.
  • Relevant parts of the deletion chromosomes from patients 3214 and 3824 are shown as stippled lines.
  • Probes used to characterize the NotI fragment and the deletions, and three YACs from which subclones were obtained, are shown below the restriction map.
  • the chimeric end of YAC 183H12 is indicated by a dotted line.
  • the orientation and approximate position of MCC are indicated above the map.
  • Figure 6 shows the DNA sequence and predicted amino acid sequence of DPI (TB2). The nucleotide numbering begins at the most 5' nucleotide isolated. A proposed initiation methionine (base 77) is indicated in bold type. The entire coding sequence is presented.
  • Figure 7 shows the cDNA and predicted amino acid sequence of DP2.5 (APC).
  • the nucleotide numbering begins at the proposed initiation methionine.
  • the nucleotides and amino acids of the alternatively spliced exon (exon 9) are at nucleotide positions 934-1236.
  • a poly(A) addition signal occurs at 9530, and one cDNA clone has a poly(A) at 9563.
  • Other cDNA clones extend beyond 9563, however, and their consensus sequence is included here.
  • Figure 8 shows the arrangement of exons in DP2.5 (APC).
  • Exon 9 corresponds to nucleotides 933-1312; exon 9a corresponds to nucleotides 1236-1312.
  • the stop codon in the cDNA is at nucleotide 8535.
  • B Partial intronic sequence surrounding each exon is shown.
  • APC Adomatous Polyposis Coli
  • alteration of the wild-type APC gene is detected.
  • "Alteration of a wild-type gene” according to the present invention encompasses all forms of mutations - including deletions. The alteration may be due to either rearrangements such as insertions, inversions, and deletions, or to point mutations. Deletions may be of the entire gene or only a portion of the gene. Somatic mutations are those which occur only in certain tissues, e.g., in the tumor tissue, and are not inherited in the germline. Germline mutations can be found in any of a body's tissues. If only a single allele is somatically mutated, an early neoplastic state is indicated.
  • APC allele which is not deleted can be screened for other mutations, such as insertions, small deletions, and point mutations. It is believed that many mutations found in tumor tissues will be those leading to decreased expression of the APC gene product. However, mutations leading to non-functional gene products would also lead to a cancerous state. Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the APC gene product.
  • tissue In order to detect the alteration of the wild-type APC gene in a tissue, it is helpful to isolate the tissue free from surrounding normal tissues.
  • Means for enriching a tissue preparation for tumor cells are known in the art.
  • the tissue may be isolated from paraffin or cryostat sections. Cancer cells may also be separated from normal cells by flow cytometry. These as well as other techniques for separating tumor from normal cells are well known in the art. If the tumor tissue is highly contaminated with normal cells, detection of mutations is more difficult.
  • Detection of point mutations may be accomplished by molecular cloning of the APC allele (or alleles) and sequencing that allele(s) using techniques well known in the art.
  • the polymerase chain reaction PCR
  • PCR polymerase chain reaction
  • the polymerase chain reaction itself is well known in the art. See, e.g., Saiki et al., Science, Vol. 239, p. 487, 1988; U.S. 4,683,203; and U.S. 4,683,195.
  • Specific primers which can be used in order to amplify the gene will be discussed in more detail below.
  • the ligase chain reaction which is known in the art, can also be used to amplify APC sequences. See Wu et al., Genomics, Vol. 4, pp. 560-569 (1989).
  • a technique known as allele specific PCR can be used. (See Ruano and Kidd, Nucleic Acids Research, Vol. 17, p. 8392, 1989.)
  • primers are used which hybridize at their 3' ends to a particular APC mutation. If the particular APC mutation is not present, an amplification product is not observed.
  • Amplification Refractory Mutation System (ARMS) can also be used as disclosed in European Patent Application Publication No. 0332435 and in Newton et al., Nucleic Acids Research, Vol.
  • Insertions and deletions of genes can also be detected by cloning, sequencing and amplification.
  • restriction fragment length polymorphism (RFLP) probes for the gene or surrounding marker genes can be used to score alteration of an allele or an insertion in a polymorphic fragment.
  • RFLP restriction fragment length polymorphism
  • Such a method is particularly useful for screening among kindred persons of an affected individual for the presence of the APC mutation found in that individual.
  • Single stranded conformation polymorphism (SSCP) analysis can also be used to detect base change variants of an allele.
  • SSCP Single stranded conformation polymorphism
  • Other techniques for detecting insertions and deletions as are known in the art can be used.
  • Alteration of wild-type genes can also be detected on the basis of the alteration of a wild-type expression product of the gene.
  • Such expression products include both the APC mRNA as well as the APC protein product.
  • the sequences of these products are shown in Figures 3 and 7.
  • Point mutations may be detected by amplifying and sequencing the mRNA or via molecular cloning of cDNA made from the mRNA.
  • the sequence of the cloned cDNA can be determined using DNA sequencing techniques which are well known in the art.
  • the cDNA can also be sequenced via the polymerase chain reaction (PCR) which will be discussed in more detail below.
  • PCR polymerase chain reaction
  • Mismatches are hybridized nucleic acid duplexes which are not 100% homologous.
  • the lack of total homology may be due to deletions, insertions, inversions, substitutions or frameshift mutations.
  • Mismatch detection can be used to detect point mutations in the gene or its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of tumor samples.
  • An example of a mismatch cleavage technique is the RNase protection method, which is described in detail in Winter et al., Proc. Natl. Acad. Sci. USA, Vol. 82, p. 7575, 1985 and Meyers et al., Science, Vol. 230, p. 1242, 1985.
  • the method involves the use of a labeled riboprobe which is complementary to the human wild-type APC gene coding sequence.
  • the riboprobe and either mRNA or DNA isolated from the tumor tissue are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch.
  • RNA product when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is smaller than the full-length duplex RNA for the riboprobe and the mRNA or DNA.
  • the riboprobe need not be the full length of the APC mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the APC mRNA or gene it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches.
  • DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Cotton et al., Proc. Natl. Acad. Sci. USA, Vol. 85, 4397, 1988; and Shenk et al., Proc. Natl. Acad. Sci. USA, Vol. 72, p. 989, 1975.
  • mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, e.g., Cariello, Human Genetics, Vol. 42, p. 726, 1988.
  • the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridization. Changes in DNA of the APC gene can also be detected using Southern hybridization, especially if the changes are gross rearrangements, such as deletions and insertions.
  • DNA sequences of the APC gene which have been amplified by use of polymerase chain reaction may also be screened using allele-specific probes.
  • These probes are nucleic acid oligomers, each of which contains a region of the APC gene sequence harboring a known mutation. For example, one oligomer may be about 30 nucleotides in length, corresponding to a portion of the APC gene sequence.
  • PCR amplification products can be screened to identify the presence of a previously identified mutation in the APC gene.
  • Hybridization of allele-specific probes with amplified APC sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under stringent hybridization conditions indicates the presence of the same mutation in the tumor tissue as in the allele-specific probe.
  • Alteration of APC mRNA expression can be detected by any technique known in the art. These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA expression indicates an alteration of the wild-type APC gene.
  • Alteration of wild-type APC genes can also be detected by screening for alteration of wild-type APC protein.
  • monoclonal antibodies immunoreactive with APC can be used to screen a tissue. Lack of cognate antigen would indicate an APC mutation.
  • Antibodies specific for products of mutant alleles could also be used to detect mutant APC gene product.
  • immunological assays can be done in any convenient format known in the art. These include Western blots, immunohistochemical assays and ELISA assays. Any means for detecting an altered APC protein can be used to detect alteration of wild-type APC genes. Functional assays can be used, such as protein binding determinations.
  • APC protein oligomerizes to itself and/or MCC protein or binds to a G protein.
  • an assay for the ability to bind to wild type APC or MCC protein or that G protein can be employed.
  • assays can be used which detect APC biochemical function. It is believed that APC is involved in phospholipid metabolism. Thus, assaying the enzymatic products of the involved phospholipid metabolic pathway can be used to determine APC activity. Finding a mutant APC gene product indicates alteration of a wild-type APC gene.
  • Mutant APC genes or gene products can also be detected in other human body samples, such as, serum, stool, urine and sputum.
  • the same techniques discussed above for detection of mutant APC genes or gene products in tissues can be applied to other body samples. Cancer cells are sloughed off from tumors and appear in such body samples.
  • the APC gene product itself may be secreted into the extracellular space and found in these body samples even in the absence of cancer cells. By screening such body samples, a simple early diagnosis can be achieved for many types of cancers. In addition, the progress of chemotherapy or radiotherapy can be monitored more easily by testing such body samples for mutant APC genes or gene products.
  • the methods of diagnosis of the present invention are applicable to any tumor in which APC has a role in tumorigenesis.
  • Deletions of chromosome arm 5q have been observed in tumors of lung, breast, colon, rectum, bladder, liver, sarcomas, stomach and prostate, as well as in leukemias and lymphomas. Thus these are likely to be tumors in which APC has a role.
  • the diagnostic method of the present invention is useful for clinicians so that they can decide upon an appropriate course of treatment. For example, a tumor displaying alteration of both APC alleles might suggest a more aggressive therapeutic regimen than a tumor displaying alteration of only one APC allele.
  • the primer pairs of the present invention are useful for determination of the nucleotide sequence of a particular APC allele using the polymerase chain reaction.
  • the pairs of single stranded DNA primers can be annealed to sequences within or surrounding the APC gene on chromosome 5q in order to prime amplifying DNA synthesis of the APC gene itself.
  • a complete set of these primers allows synthesis of all of the nucleotides of the APC gene coding sequences, i.e., the exons.
  • the set of primers preferably allows synthesis of both intron and exon sequences. Allele specific primers can also be used. Such primers anneal only to particular APC mutant alleles, and thus will only amplify a product in the presence of the mutant allele as a template.
  • primers may have restriction enzyme site sequences appended to their 5' ends.
  • all nucleotides of the primers are derived from APC sequences or sequences adjacent to APC except the few nucleotides necessary to form a restriction enzyme site.
  • restriction enzyme site sequences are well known in the art.
  • the primers themselves can be synthesized using techniques which are well known in the art. Generally, the primers can be made using oligonucleotide synthesizing machines which are commercially available. Given the sequence of the APC open reading frame shown in Figure 7, design of particular primers is well within the skill of the art.
  • the nucleic acid probes provided by the present invention are useful for a number of purposes. They can be used in Southern hybridization to genomic DNA and in the RNase protection method for detecting point mutations already discussed above.
  • the probes can be used to detect PCR amplification products. They may also be used to detect mismatches with the APC gene or mRNA using other techniques. Mismatches can be detected using either enzymes (e.g., S1 nuclease), chemicals (e.g., hydroxylamine or osmium tetroxide and piperidine), or changes in electrophoretic mobility of mismatched hybrids as compared to totally matched hybrids. These techniques are known in the art.
  • probes are complementary to APC gene coding sequences, although probes to certain introns are also contemplated.
  • An entire battery of nucleic acid probes is used to compose a kit for detecting alteration of wild-type APC genes. The kit allows for hybridization to the entire APC gene. The probes may overlap with each other or be contiguous.
  • a riboprobe is used to detect mismatches with mRNA, it is complementary to the mRNA of the human wild-type APC gene.
  • the riboprobe thus is an anti-sense probe in that it does not code for the APC protein because it is of the opposite polarity to the sense strand.
  • the riboprobe generally will be labeled with a radioactive, colorimetric, or fluorometric material, which can be accomplished by any means known in the art. If the riboprobe is used to detect mismatches with DNA it can be of either polarity, sense or anti-sense. Similarly, DNA probes also may be used to detect mismatches.
  • Nucleic acid probes may also be complementary to mutant alleles of the APC gene. These are useful to detect similar mutations in other patients on the basis of hybridization rather than mismatches. These are discussed above and referred to as allele-specific probes.
  • the APC probes can also be used in Southern hybridizations to genomic DNA to detect gross chromosomal changes such as deletions and insertions.
  • the probes can also be used to select cDNA clones of APC genes from tumor and normal tissues.
  • the probes can be used to detect APC mRNA in tissues to determine if expression is diminished as a result of alteration of wild-type APC genes.
  • Figure 7 SEQ ID NO: 1
  • a method is also provided of supplying wild-type APC function to a cell which carries mutant APC alleles. Supplying such function should suppress neoplastic growth of the recipient cells.
  • the wild-type APC gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal. In such a situation the gene will be expressed by the cell from the extrachromosomal location. If a gene portion is introduced and expressed in a cell carrying a mutant APC allele, the gene portion should encode a part of the APC protein which is required for non-neoplastic growth of the cell.
  • the wild-type APC gene or a part of it is introduced into the mutant cell in such a way that it recombines with the endogenous mutant APC gene present in the cell.
  • Such recombination requires a double recombination event which results in the correction of the APC gene mutation.
  • Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art and any suitable vector may be used.
  • Methods for introducing DNA into cells such as electroporation, calcium phosphate co-precipitation and viral transduction are known in the art and the choice of method is within the competence of the routineer.
  • Cells transformed with the wild-type APC gene can be used as model systems to study cancer remission and drug treatments which promote such remission.
  • cells and animals which carry a mutant APC allele can be used as model systems to study and test for substances which have potential as therapeutic agents.
  • the cells are typically cultured epithelial cells. These may be isolated from individuals with APC mutations, either somatic or germline. Alternatively, the cell line can be engineered to carry the mutation in the APC allele.
  • the neoplastically transformed phenotype of the cell will be determined. Any trait of neoplastically transformed cells can be assessed, including anchorage-independent growth, tumorigenicity in nude mice, invasiveness of cells, and growth factor dependence. Assays for each of these traits are known in the art.
  • Animals for testing therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. Such treatments include insertion of mutant APC alleles, usually from a second animal species, as well as insertion of disrupted homologous genes. Alternatively, the endogenous APC gene(s) of the animals may be disrupted by insertion or deletion mutation.
  • test substances After test substances have been administered to the animals, the growth of tumors must be assessed. If the test substance prevents or suppresses the growth of tumors, then the test substance is a candidate therapeutic agent for the treatment of FAP and/or sporadic cancers.
  • Polypeptides which have APC activity can be supplied to cells which carry mutant or missing APC alleles.
  • the sequence of the APC protein is disclosed in Figure 3 or 7 (SEQ ID NO: 7 or 1). These two sequences differ slightly and appear to be indicate the existence of two different forms of the APC protein.
  • Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors.
  • APC can be extracted from APC-producing mammalian cells such as brain cells.
  • the techniques of synthetic chemistry can be employed to synthesize APC protein. Any of such techniques can provide the preparation of the present invention which comprises the APC protein. The preparation is substantially free of other human proteins. This is most readily accomplished by synthesis in a microorganism or in vitro.
  • Active APC molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some such active molecules may be taken up by cells, actively or by diffusion. Extracellular application of APC gene product may be sufficient to affect tumor growth. Supply of molecules with APC activity should lead to a partial reversal of the neoplastic state. Other molecules with APC activity may also be used to effect such a reversal, for example peptides, drugs, or organic compounds.
  • the present invention also provides a preparation of antibodies immunoreactive with a human APC protein.
  • the antibodies may be polyclonal or monoclonal and may be raised against native APC protein, APC fusion proteins, or mutant APC proteins.
  • the antibodies should be immunoreactive with APC epitopes, preferably epitopes not present on other human proteins.
  • the antibodies will immunoprecipitate APC proteins from solution as well as react with APC protein on Western or immunoblots of polyacrylamide gels.
  • the antibodies will detect APC proteins in paraffin or frozen tissue sections, using immunocytochemical techniques. Techniques for raising and purifying antibodies are well known in the art and any such techniques may be chosen to achieve the preparation of the invention.
  • Predisposition to cancers as in FAP and GS can be ascertained by testing any tissue of a human for mutations of the APC gene. For example, a person who has inherited a germline APC mutation would be prone to develop cancers. This can be determined by testing DNA from any tissue of the person's body. Most simply, blood can be drawn and DNA extracted from the cells of the blood. In addition, prenatal diagnosis can be accomplished by testing fetal cells, placental cells, or amniotic fluid for mutations of the APC gene. Alteration of a wild-type APC allele, whether for example, by point mutation or by deletion, can be detected by any of the means discussed above.
  • Molecules of cDNA according to the present invention are intron-free, APC gene coding molecules. They can be made by reverse transcriptase using the APC mRNA as a template. These molecules can be propagated in vectors and cell lines as is known in the art. Such molecules have the sequence shown in SEQ ID NO: 7.
  • the cDNA can also be made using the techniques of synthetic chemistry given the sequence disclosed herein.
  • a short region of homology has been identified between APC and the human m3 muscarinic acetylcholine receptor (mAChR). This homology was largely confined to 29 residues in which 6 out of 7 amino acids (EL(GorA)GLQA) were identical (See Figure 4). Initially, it was not known whether this homology was significant, because many other proteins had higher levels of global homology (though few had six out of seven contiguous amino acids in common). However, a study on the sequence elements controlling G protein activation by mAChR subtypes (Lechleiter et al., EMBO J., p.
  • This agent has been shown to inhibit the growth of benign colon tumors in patients with FAP, presumably by virtue of its activity as a cyclooxygenase inhibitor (Waddell et al., J. Surg. Oncology 24(1), 83 (1983); Wadell, et al., Am. J. Surg., 157(1), 175 (1989); Charneau et al., Gastroenterologie Clinique at Bitechnik 14(2), 153 (1990)). Cyclooxygenase is required to convert arachidonic acid to prostaglandins and other biologically active molecules. G proteins are known to regulate phospholipase A2 activity, which generates arachidonic acid from phospholipids (Role et al., Proc.
  • This example demonstrates the isolation of a 5.5 Mb region of human DNA linked to the FAP locus. Six genes are identified in this region, all of which are expressed in normal colon cells and in colorectal, lung, ad bladder tumors.
  • the cosmid markers YN5.64 and YN5.48 have previously been shown to delimit an 8 cM region containing the locus for FAP (Nakamura et al., Am. J. Hum. Genet. Vol. 43, p. 638 (1988)). Further linkage and pulse-field gel electrophoresis (PFGE) analysis with additional markers has shown that the FAP locus is contained within a 4 cM region bordered by cosmids EF5.44 and L5.99.
  • a yeast artificial chromosome (YAC) library was screened with various 5q21 markers. Twenty-one YAC clones, distributed within six contigs and including 5.5 Mb from the region between YN5.64 and YN5.48, were obtained ( Figure 1A).
  • chromosomal walking was performed.
  • YAC termini were isolated by a PCR based method and sequenced (Riley et al., Nucleic Acids Research, Vol. 18, p. 2887 (1990)).
  • PCR primers based on these sequences were then used to rescreen the YAC library.
  • the sequence from an intron of the FER gene (Hao et al., Mol. Cell. Biol., Vol. 9, p. 1587 (1989)) was used to design PCR primers for isolation of the 28EC1 and 5EH8 YACs.
  • the termini of the 28EC1 YAC were sequenced to derive markers RHE28 and LHE28, respectively. The sequences of these two markers were then used to isolate YAC clones 15CH12 (from RHE28) and 40CF1 and 29EF1 (from LHE28). These five YAC's formed a contig encompassing 1200 kb (contig 1, Figure 1B).
  • contig 2 was initiated using cosmid N5.66 sequences
  • contig 3 was initiated using sequences both from the MCC gene and from cosmid EF5.44.
  • a walk in the telomeric direction from YAC 14FH1 and a walk in the opposite direction from YAC 39GG3 allowed connection of the initial contig 3 clones through YAC 37HG4 ( Figure 1B).
  • Contig #1 FER - The FER gene was discovered through its homology to the viral oncogene ABL (Hao et al., supra). It has an intrinsic tyrosine kinase activity, and in situ hybridization with an FER probe showed that the gene was located at 5q11-23 (Morris et al., Cytogenet. Cell. Genet., Vol. 53, p. 4, (1990)). Because of the potential role of this oncogene-related gene in neoplasia, we decided to evaluate it further with regards to the FAP locus.
  • a human genomic clone from FER was isolated (MF 2.3) and used to define a restriction fragment length polymorphism (RFLP), and the RFLP in turn used to map FER by linkage analysis using a panel of three generation families. This showed that FER was very tightly linked to previously defined polymorphic markers for the FAP locus.
  • the genetic mapping of FER was complemented by physical mapping using the YAC clones derived from FER sequences ( Figure 1B). Analysis of YAC contig 1 showed that FER was within 600 kb of cosmid marker M5.28, which maps to within 1.5 Mb of cosmid L5.99 by PFGE of human genomic DNA. Thus, the YAC mapping results were consistent with the FER linkage data and PFGE analyses.
  • Contig 2 TB1 - TB1 was identified through a cross-hybridization approach. Exons of genes are often evolutionarily conserved while introns and intergenic regions are much less conserved. Thus, if a human probe cross-hybridizes strongly to the DNA from non-primate species, there is a reasonable chance that it contains exon sequences. Subclones of the cosmids shown in Figure 1 were used to screen Southern blots containing rodent DNA samples. A subclone of cosmid N5.66 (p 5.66-4) was shown to strongly hybridize to rodent DNA, and this clone was used to screen cDNA libraries derived from normal adult colon and fetal liver.
  • TB1 conserved regions of TB1 (underlined in Figure 2A) may define a predictive motif for this sequence family.
  • TB1 appeared to contain a signal peptide (or mitochondrial targeting sequence) as well as at least 7 transmembrane domains.
  • MCC MCC, TB2, SRP and APC -
  • the MCC gene was also discovered through a cross-hybridization approach, as described previously (Kinzler et al., Science Vol. 251, p. 1366 (1991)).
  • the MCC gene was considered a candidate for causing FAP by virtue of its tight genetic linkage to FAP susceptibility and its somatic mutation in sporadic colorectal carcinomas.
  • mapping experiments suggested that the coding region of MCC was approximately 50 kb proximal to the centromeric end of a 200 kb deletion found in an FAP patient.
  • MCC cDNA probes detected a 10 kb mRNA transcript on Northern blot analysis of which 4151 bp, including the entire open reading frame, have been cloned. Although the 3' non-translated portion or an alternatively spliced form of MCC might have extended into this deletion, it was possible that the deletion did not affect the MCC gene product. We therefore used MCC sequences to initiate a YAC contig, and subsequently used the YAC clones to identify genes 50 to 250 kb distal to MCC that might be contained within the deletion.
  • YS11 Another clone identified through the YAC 24ED6 screen appeared to contain portions of two distinct genes. Sequences from one end of YS11 were identical to at least 180 bp of the signal recognition particle protein SRP19 (Lingelbach et al. Nucleic Acids Research, Vol. 16, p. 9431 (1988). A second ORF, from the opposite end of clone YS11, proved to be identical to 78 bp of a novel gene which was independently identified through a second YAC-based approach. For the latter, DNA from yeast cells containing YAC 14FH1 ( Figure 1B) was digested with EcoRI and subcloned into a plasmid vector.
  • Plasmids that contained human DNA fragments were selected by colony hybridization using total human DNA as a probe. These clones were then used to search for cross-hybridizing sequences as described above for TB1, and the cross-hybridizing clones were subsequently used to screen cDNA libraries.
  • One of the cDNA clones discovered in this way (FH38) contained a long ORF (2496 bp), 78 bp of which were identical to the above-noted sequences in YS11. The ends of the FH38 cDNA clone were then used to initiate cDNA walking to extend the sequence.
  • APC cDNA clones
  • APC cDNA clones hybridized to a single transcript of approximately 9.5 kb, suggesting that the great majority of the gene product was represented in the cDNA clones obtained.
  • Sequences from the 5' end of the APC gene were found in YAC 37HG4 but not in YAC 14FH1. However, the 3' end of the APC gene was found in 14FH1 as well as 37HG4.
  • the yeast artificial chromosome of the present invention designated YAC 37HG4 has been deposited with the National Collection of Industrial and Marine Bacteria (NCIMB), P.O. Box 31, 135 Abbey Road, Aberdeen AB9 8DG, Scotland, prior to the filing of this patent application.
  • NCIMB Accession Number of YAC clone YAC 37HG4 is 40353.
  • the 5' end of the MCC coding region was found in YAC clones 19AA9 and 26GC3 but not 24ED6 or 14FH1, while the 3' end displayed the opposite pattern.
  • MCC and APC transcription units pointed in opposite directions, with the direction of transcription going from centromeric to telomeric in the case of MCC, and telomeric to centromeric in the case of APC.
  • PFGE analysis of YAC DNA digested with various restriction endonucleases showed that TB2 and SRP were between MCC and APC, and that the 3' ends of the coding regions of MCC and APC were separated by approximately 150 kb ( Figure 1B).
  • the N-terminal 25% of the protein had a high content of leucine residues (12%) and showed local sequence similarities to myosins, various intermediate filament proteins (e.g., desmin, vimentin, neurofilaments) and Drosophila armadillo/human plakoglobin.
  • the latter protein is a component of adhesive junctions (desmosomes) joining epithelial cells (Franke et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 86, p. 4027 (1989); Perfer et al., Cell, Vol. 63, p.
  • APC APC 1167 (1990)
  • the C-terminal 75% of APC is 17% serine by composition with serine residues more or less uniformly distributed. This large domain also contains local concentrations of charged (mostly acidic) and proline residues. There was no indication of potential signal peptides, transmembrane regions, or nuclear targeting signals in APC, suggesting a cytoplasmic localization.
  • MCC has the potential to form alpha helical coiled coils (Nature, Vol. 351, p. 188 (1991).
  • Lupas and colleagues have recently developed a program for predicting coiled coil potential from primary sequence data (Science, Vol. 252, p. 1162 (1991) and we have used their program to analyze both MCC and APC.
  • Analysis of MCC indicated a discontinuous pattern of coiled-coil domains separated by putative "hinge” or "spacer” regions similar to those seen in laminin and other intermediate filament proteins.
  • APC sequence revealed two regions in the N-terminal domain which had strong coiled coil-forming potential, and these regions corresponded to those that showed local similarities with myosin and IF proteins on database searching.
  • one other putative coiled coil region was identified in the central region of APC.
  • the potential for both APC and MCC to form coiled coils is interesting in that such structures often mediate homo- and hetero-oligomerization.
  • MCC shared a short similarity with the region of the m3 muscarinic acetylcholine receptor (mAChR) known to regulate specificity of G-protein coupling.
  • the APC gene also contained a local similarity to the region of the m3 mAChR that overlapped with the MCC similarity ( Figure 4B).
  • Figure 4A the similarities to ral2 ( Figure 4A) and m3 mAChR ( Figure 4B) were not statistically significant, they were interesting in light of previous observations relating G-proteins to neoplasia.
  • Each of the six genes described above was expressed in normal colon mucosa, as indicated by their representation in colon cDNA libraries.
  • PCR reverse transcription-polymerase chain reaction
  • Primers based on the sequences of FER, TB1, TB2, MCC, and APC were each used to design primers for PCR performed with cDNA templates.
  • Each of these genes was found to be expressed in normal colon, in each of ten cell lines derived from colorectal cancers, and in tumor cell lines derived from lung and bladder tumors.
  • the ten colorectal cancer cell lines included eight from patients with sporadic CRC and two from patients with FAP.
  • This example demonstrates a genetic analysis of the role of the FER gene in FAP and sporadic colorectal cancers.
  • FER a candidate because of its proximity to the FAP locus as judged by physical and genetic criteria (see Example 1), and its homology to known tyrosine kinases with oncogenic potential.
  • Primers were designed to PCR-amplify the complete coding sequence of FER from the RNA of two colorectal cancer cell lines derived from FAP patients.
  • cDNA was generated from RNA and used as a template for PCR.
  • the primers used were 5'-AGAAGG A TC C CTTGTGCAGTGTGGA-3' and 5'-GACAG GA TCCTGAAGCTGAGTTTG-3'.
  • the underlined nucleotides were altered from the true FER sequence to create BamHI sites.
  • the cell lines used were JW and Difi, both derived from colorectal cancers of FAP patients. (C. Paraskeva, B.G. Buckle, D. Sheer, C.B. Wigley, Int. J. Cancer 34, 49 (1984); M.E. Gross et al., Cancer Res. 51, 1452 (1991).
  • the resultant 2554 basepair fragments were cloned and sequenced in their entirety.
  • the PCR products were cloned in the BamHI site of Bluescript SK (Stratagene) and pools of at least 50 clones were sequenced en masse using T7 polymerase, as described in Nigro et al., Nature 342, 705 (1989).
  • PCR products were digested with PstI, which yields a 50 bp fragment if codon 439 is leucine, but 26 and 24 bp fragments if it is valine.
  • the primers used for sequencing were chosen from the FER cDNA sequence in Hao et al., supra.
  • nt 1956-2256 The three exons examined included those containing nt 822-930, 931-1309, and the first 300 nt of the most distal exon (nt 1956-2256). PCR and RNase protection analysis were performed as described in Kinzler et al. supra , using the primers underlined in Table I. The primers for nt 1956-2256 were 5'-GCAAATCCTAAGAGAGAACAA-3' and 5'-GATGGCAAGCTTGAGCCAG-3'.
  • the RNase protection method was used to screen for mutations and in an additional 13 kindreds, the PCR products were cloned and sequenced to search for mutations not detectable by RNase protection. PCR products were cloned into a Bluescript vector modified as described in T.A. Holton and M.W. Graham, Nucleic Acids Res. 19, 1156 (1991). A minimum of 100 clones were pooled and sequenced. Five variants were detected among the 103 kindreds analyzed. Cloning and subsequent DNA sequencing of the PCR product of patient P21 indicated a C to T transition in codon 413 that resulted in a change from arginine to cysteine.
  • Tumor T27 contained a somatic mutation of CGA (arginine) to TGA (stop codon) at codon 33.
  • Tumor T135 contained a GT to GC change at a splice donor site.
  • Tumor T34 contained a 5 bp insertion (CAGCC between codons 289 and 290) resulting in a stop at codon 292 due to a frameshift.
  • DNA was purified from paraffin sections as described in S.E. Goelz, S.R. Hamilton, and B. Vogelstein. Biochem. Blophys. Res. Comm. 130, 118 (1985). PCR was performed as described in reference 24, using the primers 5'-GTTCCAGCAGTGTCACAG-3' and 5'-GGGAGATTTCGCTCCTGA-3'. A PCR product containing codon 1338 was amplified from the archival DNA and used to show that the stop codon represented a somatic mutation present in the original primary tumor and in cell lines derived from the primary and metastatic tumor sites, but not from normal tissue of the patient.
  • This example characterizes small, nested deletions in DNA from two unrelated FAP patients.
  • DNA from 40 FAP patients was screened with cosmids that had been mapped into a region near the APC locus to identify small deletions or rearrangements.
  • the DNA of one FAP patient, 3214 showed only a 940 kb NotI fragment instead of the expected 1200 kb fragment.
  • DNA was analyzed from four other members of the patient's immediate family; the 940 kb fragment was present in her affected mother (4711), but not in the other, unaffected family members. The mother also carried a normal 1200 kb NotI fragment that was transmitted to her two unaffected offspring. These observations indicated that the mutant polyposis allele is on the same chromosome as the 940 kb NotI fragment.
  • a simple interpretation is that APC patients 3214 and 4711 each carry a 260 kb deletion within the APC locus.
  • the father is, therefore, interpreted as heterozygous for a polymorphism at the NotI site, with one chromosome 5 having a 1200 kb NotI fragment and the other having a fragment too large to resolve consistently on the gel. The latter was transmitted to patient 3214.
  • Probe Map30 was isolated from this library. In normal individuals probe Map30 hybridizes to the 1200 kb NotI fragment and to a 200 kb NruI fragment. This latter hybridization places Map30 distal, with respect to the locations of L5.71 and L5.79, to the NruI site of the 550 kb NotI-NruI fragment.
  • Map30 hybridized to the abnormal, 1300 kb NruI fragment of patient 3214, the locus defined by Map30 lies outside the hypothesized deletion. Furthermore, in normal chromosomes Map30 identified a 200 kb NruI fragment and L5.79 identified a 1200 kb Nrul fragment; the hypothesized deletion must, therefore, be removing an NruI site, or sites, lying between Map30 and L5.79, and these two probes must flank the hypothesized deletion. A restriction map of the genomic region, showing placement of these probes, is shown in Figure 5.
  • the 830 kb MluI fragment recognized by probe L5.79 must contain a NotI site in HHW1159 DNA; because the 860 kb MluI fragment remains intact, it does not carry this NotI site and must be distinct from the 830 kb MluI fragment.
  • Example 4 This example demonstrates the isolation of human sequences which span the region deleted in the two unrelated FAP patients characterized in Example 4.
  • YAC 57B8 (clone AT57) was found to lie within the patient 3214 deletion.
  • Inverse polymerase chain reaction (PCR) defined the end sequences of the insert of YAC 57B8.
  • PCR primers based on one of these end sequences repeatedly failed to amplify DNA from the somatic cell hybrid (HHW1155) carrying the deleted homolog of patient 3214, but did amplify a product of the expected size from the somatic cell hybrid (HHW1159) carrying the normal chromosome 5 homolog. This result supported the interpretation that the abnormal restriction fragments found in the DNA of patient 3214 result from a deletion.
  • probe Y10 hybridizes to the same 620 kb NotI-MluI fragment seen with probe L5.79 in normal DNA, indicating its location as L5.79-proximal to the deleted Mlul site and placing it between the MluI site and the L5.79-proximal NruI site.
  • the MluI site must, therefore, lie between the NruI sites that define the 150 kb NruI fragment (see Figure 5).
  • Probe Y11 also hybridized to the 150 kb NruI fragment in the normal chromosome 5 homolog, but failed to hybridize to the 620 kb NotI-MluI fragment, placing it L5.79-distal to the MluI site, but proximal to the second NruI site. Hybridization to the same (860 kb) MluI fragment as Map30 confirmed the localization of probe Y11 L5.79-distal to the MluI site.
  • Probe Y14 was shown to be L5.79-distal to both deleted NruI sites by virtue of its hybridization to the same 200 kb NruI fragment of the normal chromosome 5 seen with Map30. Therefore, the order of these EcoRI fragments derived from YAC 183H12 and deleted in patient 3214, with respect to L5.79 and Map30, is L5.79-Y10-Y11-Y14-Map30.
  • the 100 kb deletion of patient 3824 was confirmed by the failure of aberrant restriction fragments in this DNA to hybridize with probe Y11, combined with positive hybridizations to probes Y10 and/or Y14.
  • Y10 and Y14 each hybridized to the 1100 kb NotI fragment of patient 3824 as well as to the normal 1200 kb NotI fragment, but Y11 hybridized to the 1200 kb fragment only.
  • probe Y14 hybridized to the 860 kb and 760 kb fragments of patient 3824 DNA, but probe Y11 hybridized only to the 860 kb fragment.
  • probes Y10 and Y14 are missing from the deleted 3214 chromosome, but present on the deleted 3824 chromosome, and they have been shown to flank probe Y11, the deletion in patient 3824 must be nested within the patient 3214 deletion.
  • Probes Y10, Y11, Y14 and Map30 each hybridized to YAC 310D8, indicating that this YAC spanned the patient 3824 deletion and at a minimum, most of the 3214 deletion.
  • the YAC characterizations therefore, confirmed the presence of deletions in the patients and provided physical representation of the deleted region.
  • MCC An interesting FAP candidate gene, MCC, recently was ascertained with cosmid L5.71 and was shown to have undergone mutation in colon carcinomas (Kinzler et al., supra ). It was therefore of interest to map this gene with respect to the deletions in APC patients.
  • Hybridization of MCC probes with an overlapping series of YAC clones extending in either direction from L5.71 showed that the 3' end of MCC must be oriented toward the region of the two APC deletions.
  • clone 1CI contains sequences from the C-terminal end of the open reading frame, which stops at nucleotide 2708, as well as 3' untranslated sequence.
  • Clone 7 contains sequence that is entirely 3' to the open reading frame. Importantly, the entire 3' untranslated sequence contained in the cDNA clones consists of a single 2.5 kb exon. These two clones were hybridized to DNAs from the YACs spanning the FAP region.
  • Clone 7 fails to hybridize to YAC 310D8, although it does hybridize to YACs 183H12 and 57B8; the same result was obtained with the cDNA 1CI. Furthermore, these probes did show hybridization to DNAs from both hybrid cell lines (HWW1159 and HWW1155) and the lymphoblastoid cell line from patient 3214, confirming their locations outside the deleted region. Additional mapping experiments suggested that the 3' end of the MCC cDNA clone contig is likely to be located more than 45 kb from the deletion of patient 3214 and, therefore, more than 100 kb from the deletion of patient 3824.
  • This example identifies three genes within the deleted region of chromosome 5 in the two unrelated FAP patients characterized in Example 4.
  • Genomic clones were used to screen cDNA libraries in three separate experiments. One screening was done with a phage clone derived from YAC 310D8 known to span the 260 kb deletion of patient 3214. A large-insert phage library was constructed from this YAC; screening with Y11 identified ⁇ 205, which mapped within both deletions. When clone ⁇ 205 was used to probe a random-, plus oligo(dT)-, primed fetal brain cDNA library (approximately 300,000 phage), six cDNA clones were isolated and each of them mapped entirely within both deletions.
  • cDNA walks yielded a cDNA contig of 3.0-3.5 kb, and included two clones containing terminal poly(A) sequences. This size corresponds to the 3.5 kb band seen by Northern analysis. Sequencing of the first 3163 bp of the cDNA contig revealed an open reading frame extending from the first base to nucleotide 631, followed by a 2.5 kb 3' untranslated region. The sequence surrounding the methionine codon at base 77 conforms to the Kozak consensus of an initiation methionine (Kozak, 1984). Failed attempts to walk farther, coupled with the similarity of the lengths of isolated cDNA and mRNA, suggested that the NH 2 -terminus of the DPI protein had been reached. Hybridization to a combination of genomic and YAC DNAs cut with various enzymes indicated the genomic coverage of DPI to be approximately 30 kb.
  • YS-11 and YS-39 Two additional probes for the locus, YS-11 and YS-39, which had been ascertained by screening of a cDNA library with an independent YAC probe identified with MCC sequences adjacent to L5.71, were mapped into the deletion region.
  • YS-39 was shown to be a cDNA identical in sequence to DPI. Partial characterization of YS-11 had shown that 200 bp of DNA sequence at one end was identical to sequence coding for the 19 kd protein of the ribosomal signal recognition particle, SRP19 (Lingelbach et al., supra ). Hybridization experiments mapped YS-11 within both deletions. The sequence of this clone, however, was found to be complex.
  • YS-11 was either a chimeric clone containing two independent inserts or a clone of an incompletely processed or aberrant message. If YS-11 were a conventional chimeric clone, the independent segments would not be expected to map to the same physical region. The segments resulting from anomalous processing of a continuous transcript, however, would map to a single chromosomal region.
  • V5 Of the 14 cDNA clones selected from the fetal brain library, one clone, V5, was of particular interest in that it contained an open reading frame throughout, although it included only a short identity to the first 78 5' bases of the YS-11 sequence. Following the 78 bp of identical sequence, the two cDNA sequences diverged at an AG. Furthermore, divergence from genomic sequence was also seen after these 78 bp, suggesting the presence of a splice junction, and supporting the view that YS-11 represents an irregular message.
  • the third screening approach relied on hybridization with a 120 kb MluI fragment from YAC 57B8. This fragment hybridizes with probe Y11 and completely spans the 100 kb deletion in patient 3824. the fragment was purified on two preparative PFGs, labeled, and used to screen a fetal brain cDNA library. A number of cDNA clones previously identified in the development of the DPI and DP2 contigs were reascertained. However, 19 new cDNA clones mapped into the patient 3824 deletion. Analysis indicated that these 19 formed a new contig, DP3, containing a large open reading frame.
  • the consensus cDNA sequence of DP2.5 suggests that the entire coding sequence of DP2.5 has been obtained and is 8532 bp long. The most 5' ATG codon occurs two codons from an in-frame stop and conforms to the Kozak initiation consensus (Kozak, Nucl. Acids. Res., Vol. 12, p. 857-872 1984). The 3' open reading frame breaks down over the final 1.8 kb, giving multiple stops in all frames. A poly(A) sequence was found in one clone approximately 1 kb into the 3' untranslated region, associated with a polyadenylation signal 33 bp upstream (position 9530). The open reading frame is almost identical to that identified as APC above.
  • nucleotide 934 of the DP2.5 transcript is of potential interest. It was first discovered by noting that two classes of cDNA had been isolated. The more abundant cDNA class contains a 303 bp exon not included in the other. The presence in vivo of the two transcripts was verified by an exon connection experiment. Primers flanking the alternatively spliced exon were used to amplify, by PCR, cDNA prepared from various adult tissues. Two PCR products that differed in size by approximately 300 bases were amplified from all the tissues tested; the larger product was always more abundant than the smaller.
  • This example demonstrates the primers used to identify subtle mutations in DP1, SRP19, and DP25.
  • sequencing substrate was obtained by inverse PCR amplification of DNAs from two YACs, 310D8 and 183H12, that span the deletions. Ligation at low concentration cyclized the restriction enzyme-digested YAC DNAs. Oligonucleotides with sequencing tails, designed in inverse orientation at intervals along the cDNAs, primed PCR amplification from the cyclized templates. Comparison of these DNA sequences with the cDNA sequences placed exon boundaries at the divergence points. SRP19 and DPI were each shown to have five exons. DP2.5 consisted of 15 exons.
  • the sequences of the oligonucleotides synthesized to provide PCR amplification primers for the exons of each of these genes are listed in Table III. With the exception of exons 1, 3, 4, 9, and 15 of DP2.5 (see below), the primer sequences were located in intron sequences flanking the exons.
  • the 5' primer of exon 1 is complementary to the cDNA sequence, but extends just into the 5' Kozak consensus sequence for the initiator methionine, allowing a survey of the translated sequences.
  • the 5' primer of exon 3 is actually in the 5' coding sequences of this exon, as three separate intronic primers simply would not amplify.
  • the 5' primer of exon 4 just overlaps the 5' end of this exon, and we thus fail to survey the 19 most 5' bases of this exon.
  • two overlapping primer sets were used, such that each had one end within the exon.
  • the large 3' exon of DP2.5 overlapping primer pairs were placed along the length of the exon; each pair amplified a product of 250-400 bases.
  • SSCP single stranded conformation polymorphism
  • SSCP analysis identifies most single- or multiple-base changes in DNA fragments up to 400 bases in length. Sequence alterations are detected as shifts in electrophoretic mobility of single-stranded DNA on nondenaturing acrylamide gels; the two complementary strands of a DNA segment usually resolve as two SSCP conformers of distinct mobilities. However, if the sample is from an individual heterozygous for a base-pair variant within the amplified segment, often three or more bands are seen. In some cases, even the sample from a homozygous individual will show multiple bands. Base-pair-change variants are identified by differences in pattern among the DNAs of the sample set.
  • Exons of the candidate genes were amplified by PCR from the DNAs of 61 unrelated FAP patients and a control set of 12 normal individuals.
  • the five exons from DPI revealed no unique conformers in the FAP patients, although common conformers were observed with exons 2 and 3 in some individuals of both affected and control sets, indicating the presence of DNA sequence polymorphisms.
  • none of the five exons of SRP19 revealed unique conformers in DNA from FAP patients in the test panel.
  • the five sets of conformers unique to the FAP patients were sequenced to determine the nucleotide changes responsible for their altered mobilities.
  • the normal conformers from the host individuals were sequenced also. Bands were cut from the dried acrylamide gels, and the DNA was eluted. PCR amplification of these DNAs provided template for sequencing.
  • the sequences of the unique conformers from exons 7, 8, 10, and 11 of DP2.5 revealed dramatic mutations in the DP2.5 gene.
  • the sequence of the new mutation creating the exon 7 conformer in patient 3746 was shown to contain a deletion of two adjacent nucleotides, at positions 730 and 731 in the cDNA sequence ( Figure 7).
  • the normal sequence at this splice junction is CAG GGTCA (intronic sequence underlined), with the intron-exon boundary between the two repetitions of AG.
  • the mutant allele in this patient has the sequence CAGGTCA.
  • this change is at the 5' splice site, comparison with known consensus sequences of splice junctions would suggest that a functional splice junction is maintained. If this new splice junction were functional, the mutation would introduce a frameshift that creates a stop codon 15 nucleotides downstream. If the new splice junction were not functional, messenger processing would be significantly altered.
  • the PCR product from FAP patient 3746 and a control DNA were electrophoresed on an acrylamide-urea denaturing gel, along with the products of a sequencing reaction.
  • the sample from patient 3746 showed two bands differing in size by 2 nucleotides, with the larger band identical in mobility to the control sample; this result was independent confirmation that patient 3746 is heterozygous for a 2 bp deletion.
  • the conformer unique to FAP patient 3827 in exon 10 was found to contain a deletion of one nucleotide (1367, 1368, or 1369) when compared to the normal sequence found in the other bands on the SSCP gel.
  • This deletion occurring within a set of three T's, changed the sequence from CTTTCA to CTTCA; this 1 base frameshift creates a downstream stop within 30 bases.
  • the PCR product amplified from this patient's DNA also was electrophoresed on an acrylamide-urea denaturing gel, along with the PCR product from a control DNA and products from a sequencing reaction.
  • the patient's PCR product showed two bands differing by 1 bp in length, with the larger identical in mobility to the PCR product from the normal DNA; this result confirmed the presence of a 1 bp deletion in patient 3827.
  • Sequence analysis of the variant conformer of exon 11 from patient 3712 revealed the substitution of a T by a G at position 1500, changing the normal tyrosine codon to a stop codon.
  • Patient 3746 described above as carrying an APC allele with a frameshift mutation, is an affected offspring of two normal parents. Colonoscopy revealed no polyps in either parent nor among the patient's three siblings.
  • This example demonstrates polymorphisms in the APC gene which appear to be unrelated to disease (FAP).
  • Sequencing of variant conformers found among controls as well as individuals with APC has revealed the following polymorphisms in the APC gene: first, in exon 11, at position 1458, a substitution of T to C creating an RsaI restriction site but no amino acid change; and second, in exon 15, at positions 5037 and 5271, substitutions of A to G and G to T, respectively, neither resulting in amino acid substitutions.
  • These nucleotide polymorphisms in the APC gene sequence may be useful for diagnostic purposes.
  • This example shows the structure of the APC gene.
  • the structure of the APC gene is schematically shown in Figure 8, with flanking intron sequences indicated.
  • This example demonstrates the mapping of the FAP deletions with respect to the APC exons.
  • Somatic cell hybrids carrying the segregated chromosomes 5 from the 100 kb (HHW1291) and 260 kb (HHW1155) deletion patients were used to determine the distribution of the APC genes exons across the deletions. DNAs from these cell lines were used as template, along with genomic DNA from a normal control, for PCR-based amplification of the APC exons.
  • PCR analysis of the hybrids from the 260 kb deletion of patient 3214 showed that all but one (exon 1) of the APC exons are removed by this deletion.
  • This example demonstrates the expression of alternately spliced APC messenger in normal tissues and in cancer cell lines.
  • Tissues that express the APC gene were identified by PCR amplification of cDNA made to mRNA with primers located within adjacent APC exons.
  • PCR primers that flank the alternatively spliced exon 9 were chosen so that the expression pattern of both splice forms could be assessed.
  • All tissue types tested (brain, lung, aorta, spleen, heart, kidney, liver, stomach, placenta, and colonic mucosa) and cultured cell lines (lymphoblasts, HL60, and choriocarcinoma) expressed both splice forms of the APC gene.
  • lymphocytes normally residing in some tissues, including colon, prevents unequivocal assessment of expression.
  • the large mRNA, containing the complete exon 9 rather than only exon 9A appears to be the more abundant message.
  • This example discusses structural features of the APC protein predicted from the sequence.
  • the cDNA consensus sequence of APC predicts that the longer, more abundant form of the message codes for a 2842 or 28444 amino acid peptide with a mass of 311.8 kd.
  • This predicted APC peptide was compared with the current data bases of protein and DNA sequences using both Intelligenetics and GCG software packages. No genes with a high degree of amino acid sequence similarity were found. Although many short (approximately 20 amino acid) regions of sequence similarity were uncovered, none was sufficently strong to reveal which, if any, might represent functional homology. Interestingly, multiple similarities to myosins and keratins did appear.
  • the APC gene also was scanned for sequence motifs of known function; although multiple glycosylation, phosphorylation, and myristoylation sites were seen, their significance is uncertain.
  • exon 9A the deleted form of exon 9, two heptad repeat regions are reconnected in the proper heptad repeat frame, deleting the intervening peptide region. After the first 1000 residues, the high proline content of the remainder of the peptide suggests a compact rather than a rod-like structure.
  • residues 2200-24000 contain a 200 amino acid basic domain.

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Claims (40)

  1. Procédé pour diagnostiquer ou pronostiquer un tissu néoplasique d'un humain, comportant l'étape consistant à : détecter une altération somatique de séquences d'un gène APC du type sauvage ou de leurs produits d'expression dans un tissu tumoral isolé à partir d'un humain, ladite altération indiquant une néoplasie du tissu, dans lequel, en particulier, les produits d'expression sont des molécules d'ARNm, et dans lequel, en particulier, les produits d'expression sont des molécules protéiniques.
  2. Procédé selon la revendication 1, dans lequel l'altération de l'ARNm d'un gène APC du type sauvage est détectée par hybridation de l'ARNm provenant dudit tissu à une sonde d'un gène APC.
  3. Procédé selon la revendication 1, dans lequel l'altération des séquences d'un gène APC du type sauvage est détectée par une hybridation d'une sonde à séquence d'un gène APC à de l'ADN génomique isolé à partir dudit tissu, dans lequel le procédé comporte en particulier de plus les étapes consistant à :
    soumettre l'ADN génomique isolé à partir d'un tissu non-néoplasique de l'humain à une hybridation Southern avec la sonde à séquence d'un gène APC, et
    comparer les hybridations de la sonde d'un gène APC auxdits tissus tumoral et non-néoplasique ; dans lequel, en particulier, la sonde d'un gène APC détecte un polymorphisme de taille de fragments de restriction.
  4. Procédé selon la revendication 1, dans lequel la détection de l'altération de séquences d'un gène APC du type sauvage est sélectionnée parmi les techniques suivantes :
    (a) l'observation de différences de mobilité électrophorétique d'ADN simple-brin sur des gels de polyacrylamide non-dénaturants,
    (b) la détermination de la séquence de la totalité d'un gène APC, ou d'une partie de celui-ci, dans ledit tissu, des écarts dans la séquence APC déterminés par rapport à la séquence APC représentée (i) sur la figure 7 (SEQ ID n° 1), ou (ii) trouvée dans YAC 37HG4, suggérant une néoplasie,
    (c) l'identification d'une incompatibilité entre des molécules, (1) un gène APC ou un ARNm de APC isolé à partir du tissu et (2) une sonde d'acide nucléique complémentaire de la séquence d'un gène APC humain du type sauvage, lorsque les molécules (1) et (2) sont hybridées l'une à l'autre pour former un duplex,
    (d) l'amplification de séquences d'un gène APC dans ledit tissu et l'hybridation des séquences APC amplifiées à des sondes d'acide nucléique qui comportent des séquences APC,
    (e) le clonage moléculaire des gènes APC dans ledit tissu et le séquençage de la totalité du gène APC cloné, ou d'une partie de celui-ci, et
    (f) le criblage vis-à-vis d'une mutation par délétion, d'une mutation ponctuelle, d'une mutation par inversion ou d'une mutation par insertion.
  5. Procédé selon la revendication 3, dans lequel la sonde d'un gène APC s'hybride à un exon sélectionné parmi le groupe constitué de : (1) les nucléotides 822 à 930, et (2) les nucléotides 931 à 1309, (3) les nucléotides 1406 à 1545, et (4) les nucléotides 1956 à 2256.
  6. Procédé selon l'une quelconque des revendications 1 à 5, dans lequel le tissu néoplasique est un tissu colorectal, et dans lequel en particulier le tissu non-néoplasique isolé à partir d'un humain provient de la muqueuse du colon.
  7. Procédé selon la revendication 1, dans lequel la détection de l'altération de la protéine APC du type sauvage est sélectionnée parmi une immuno-cytochimie, une immuno-empreinte, une évaluation de métabolites de phospholipide, et une évaluation d'interactions de liaison entre la protéine APC dudit tissu tumoral et une seconde protéine cellulaire, dans lequel en particulier la seconde protéine cellulaire est sélectionnée parmi une protéine MCC, une protéine APC du type sauvage, et une protéine G.
  8. Procédé pour affecter in vitro une fonction d'un gène APC du type sauvage à une cellule qui a perdu ladite fonction du fait d'une mutation dans un gène APC, comportant les étapes en variante consistant à :
    introduire un gène APC du type sauvage dans une cellule qui a perdu ladite fonction génique de sorte que ledit gène APC du type sauvage est exprimé dans la cellule, en particulier le gène APC du type sauvage introduit se recombinant avec un gène APC mutant endogène présent dans la cellule par un événement de double recombinaison pour corriger la mutation du gène APC,
    introduire une partie d'un gène APC du type sauvage dans une cellule qui a perdu ladite fonction génique de sorte que ladite partie est exprimée dans la cellule, ladite partie codant pour une partie de la protéine APC qui est nécessaire à la croissance non-néoplasique de ladite cellule,
    appliquer une protéine APC humaine du type sauvage à une cellule qui a perdu la fonction d'un gène APC du type sauvage.
  9. Procédé pour affecter in vitro une fonction d'un gène APC du type sauvage à une cellule qui a une fonction d'un gène APC altérée du fait d'une mutation dans un gène APC, comportant l'introduction dans la cellule d'une molécule qui imite la fonction d'une protéine APC du type sauvage.
  10. Paire d'amorces d'ALN simple-brin pour déterminer une séquence nucléotidique d'un gène APC par réaction de polymérisation en chaíne, la séquence desdites amorces étant dérivée de la bande 21 du chromosome 5q, dans laquelle l'utilisation desdites amorces dans une réaction de polymérisation en chaíne a pour résultat la synthèse de l'ADN ayant la totalité de la séquence représentée sur la figure 7, ou une partie de celle-ci, en particulier dans laquelle les amorces ont des sites d'enzyme de restriction au niveau de chaque extrémité 5', et dans laquelle en particulier les amorces ont des séquences correspondant à des introns APC.
  11. Sonde d'acide nucléique complémentaire de séquences codant pour un gène APC humain du type sauvage, en particulier dans laquelle la sonde d'acide nucléique s'hybride à un exon sélectionné parmi le groupe constitué de : (1) les nucléotides 822 à 930, et (2) les nucléotides 931 à 1309, (3) les nucléotides 1406 à 1545, (4) les nucléotides 1956 à 2256.
  12. Kit pour détecter une altération de gènes APC du type sauvage comportant un batterie de scndes d'acide nucléique qui, dans l'agrégat, s'hybrident à tous les nucléotides des séquences codant pour un gène APC.
  13. Procédé pour détecter la présence d'un tissu néoplasique chez un humain, comportant l'étape consistant à :
       détecter dans un échantillon corporel, isolé à partir d'un humain, une altération d'une séquence d'un gène APC du type sauvage ou d'un produit d'expression d'un gène APC du type sauvage, ladite altération indiquant la présence d'un tissu néoplasique chez l'humain, dans lequel en particulier ledit échantillon corporel est sélectionné parmi un sérum, des selles, de l'urine et un crachat.
  14. Procédé pour détecter une prédisposition génétique à un cancer, incluant le polyadénome du gros intestin (FAP) et le Syndrome de Gardner (GS), chez un humain comportant l'étape consistant à :
       détecter une altération germinale de séquences d'un gène APC du type sauvage ou de leurs produits d'expression dans un échantillon humain, en particulier du sang et des tissus foetaux, ladite altération indiquant une prédisposition à un cancer.
  15. Procédé selon la revendication 14, dans lequel les produits d'expression sont des molécules d'ARNm, dans lequel en particulier l'altération de l'ARNm d'un gène APC du type sauvage est détectée par hybridation de l'ARNm provenant dudit tissu à une sonde d'un gène APC.
  16. Procédé selon la revendication 13 ou 14, dans lequel la détection de l'altération de séquences d'un gène APC du type sauvage est sélectionnée parmi les techniques suivantes :
    (a) l'observation de différences de mobilité électrophorétique d'ADN simple-brin sur des gels de polyacrylamide non-dénaturants,
    (b) l'hybridation d'une sonde à séquence d'un gène APC à de l'ADN génomique isolé à partir dudit tissu, en particulier dans lequel la sonde à séquence d'un gène APC détecte un polymorphisme de taille de fragments de restriction, en particulier dans lequel la sonde d'un gène APC s'hybride à un exon sélectionné parmi : (1) les nucléotides 822 à 930, et (2) les nucléotides 931 à 1309, (3) les nucléotides 1406 à 1545 et (4) les nucléotides 1956 à 2256,
    (c) la détermination de la séquence de la totalité d'un gène APC, ou d'une partie de celui-ci, dans ledit tissu, des écarts de séquence d'un gène APC déterminés par rapport à la séquence de la figure 7, ou la séquence trouvée dans YAC 37HG4, suggérant une prédisposition à un cancer,
    (d) l'identification d'une incompatibilité entre des molécules, (1) un gène APC ou de l'ARNm d'un gène APC isolé à partir dudit tissu et (2) une sonde d'acide nucléique complémentaire de la séquence d'un gène APC humain du type sauvage, lorsque les molécules (1) et (2) sont hybridées l'une à l'autre pour former un duplex,
    (e) l'amplification de séquences d'un gène APC dans ledit tissu et l'hybridation des séquences d'un gène APC amplifiées à des sondes d'acide nucléique qui comportent des séquences d'un gène APC,
    (f) le clonage moléculaire des gènes APC dans ledit tissu et le séquençage de la totalité du gène APC cloné, ou d'une partie de celui-ci, et
    (g) le criblage vis-à-vis d'une mutation par délétion, d'une mutation ponctuelle, d'une mutation par inversion ou un criblage vis-à-vis d'une mutation par insertion.
  17. Procédé selon la revendication 14, dans lequel les produits d'expression sont des molécules protéines, en particulier dans lequel l'altération de la protéine APC du type sauvage est détectée par immuno-empreinte, par immuno-chimie, ou par évaluation d'interactions de liaison entre la protéine APC isolée à partir dudit tissu et une seconde protéine cellulaire, en particulier dans lequel la seconde protéine cellulaire est sélectionnée parmi une protéine MCC, une protéine APC du type sauvage, et une protéine G.
  18. Procédé in vitro de criblage vis-à-vis d'une prédisposition génétique à un cancer, incluant le polyadénome du gros intestin (FAP) et le Syndrome de Gardner (GS), chez un humain comportant l'étape consistant à :
       détecter parmi des personnes apparentées la présence d'un polymorphisme d'ADN qui est lié à un allèle d'un gène APC mutant chez un individu ayant une prédisposition génétique à un cancer, ledit lien de parenté étant génétiquement associé à l'individu, la présence dudit polymorphisme suggérant une prédisposition à un cancer.
  19. Préparation de la protéine APC humaine essentiellement exempte d'autres protéines humaines, la séquence d'acides aminés de ladite protéine correspondant à celle représentée sur la figure 3 ou 7 (SEQ ID n° 1).
  20. Préparation d'anticorps immunoréactifs avec une protéine APC humaine et pratiquement non-immunoréactifs avec d'autres protéines humaines.
  21. Procédé pour tester des agents thérapeutiques vis-à-vis de la capacité à atténuer un phénotype transformé de manière néoplasique, comportant les étapes consistant à :
    appliquer une substance test à une cellule épithéliale cultivée qui porte une mutation dans un allèle APC,
    déterminer si ladite substance test atténue le phénotype transformé de manière néoplasique de la cellule, dans lequel en particulier la cellule épithéliale cultivée a été génétiquement manipulée pour porter la mutation dans l'allèle APC.
  22. Procédé pour tester des agents thérapeutiques vis-à-vis de la capacité à atténuer une croissance néoplasique, comportant les étapes consistant à :
    administrer une substance test à un animal, à l'exception d'humains, qui porte un allèle APC mutant dans son génome,
    déterminer si ladite substance test empêche ou atténue la croissance de tumeurs.
  23. Animal transgénique, à l'exception d'humains, qui porte dans son génome un allèle APC mutant provenant d'une seconde espèce animale.
  24. Animal, à l'exception d'humains, qui a été génétiquement manipulé pour contenir une mutation par insertion ou délétion qui rompt un allèle APC dans son génome.
  25. Molécule d'ADNc qui code pour une protéine APC.
  26. Molécule d'ADNc qui code pour une protéine ayant la séquence d'acides aminés représentée sur la figure 3 ou 7 (SEQ ID n° 7 ou 1).
  27. Molécule d'ADN isolée qui code pour une protéine APC.
  28. Molécule d'ADN isolée qui code pour une protéine ayant la séquence d'acides aminés représentée sur la figure 3 ou 7 (SEQ ID n° 7 ou 1).
  29. Chromosome artificiel de levure provenant du clone 37HG4 de YAC ayant le numéro de série NCIMB 40 353.
  30. Gène APC du type sauvage destiné à être utilisé dans un procédé pour affecter une fonction d'un gène APC du type sauvage à une cellule qui a perdu ladite fonction du fait d'une mutation du gène APC, dans lequel ledit gène APC du type sauvage après introduction dans la cellule est exprimé dans la cellule.
  31. Gène APC du type sauvage selon la revendication 30, dans lequel ledit gène APC du type sauvage introduit se recombine avec le gène APC mutant endogène présent dans la cellule par un événement de double recombinaison pour corriger la mutation du gène APC.
  32. Partie d'un gène APC du type sauvage destinée à être utilisée dans un procédé pour affecter une fonction d'un gène APC du type sauvage à une cellule qui a une fonction d'un gène APC altérée du fait d'une mutation dans un gène APC, dans laquelle ladite partie, après introduction dans la cellule, est exprimée dans la cellule, ladite partie codant pour une partie de la protéine APC qui est nécessaire pour une croissance non-néoplasique de ladite cellule.
  33. Protéine APC humaine du type sauvage destinée à être utilisée dans un procédé pour affecter une fonction d'un gène APC du type sauvage à une cellule qui a une fonction d'un gène APC altérée du fait d'une mutation dans un gène APC.
  34. Molécule qui imite la fonction d'une protéine APC du type sauvage destinée à être utilisée dans un procédé pour affecter une fonction d'un gène APC du type sauvage à une cellule qui a une fonction d'un gène APC altérée du fait d'une mutation dans un gène APC.
  35. Procédé selon l'une quelconque des revendications 1, 13 ou 14, dans lequel l'altération est détectée en utilisant le Système de Mutation Résistant à une Amplification (ARMS).
  36. Procédé selon l'une quelconque des revendications 1, 2, 3, 4, 13, 14, 15, 16 ou 18, dans lequel l'altération est sélectionnée parmi le groupe constitué des altérations suivantes :
    (a) transition de C en T au niveau du codon 414 ayant pour résultat un changement de Arg (CGC) en Cys (TGC),
    (b) transition de C en T au niveau du codon 302 ayant pour résultat un changement de Arg (CGA) en codon d'arrêt (TGA),
    (c) transition de C en G au niveau du codon 280 ayant pour résultat un changement de Ser (TCA) en codon d'arrêt (TGA),
    (d) transition de C en G au niveau du codon 713 ayant pour résultat un changement de Ser (TCA) en codon d'arrêt (TGA),
    (e) transition de C en T au niveau du codon 332 ayant pour résultat un changement de Arg (CGA) en codon d'arrêt (TGA),
    (f) insertion de 5 paires de bases (CAGCC) entre les codons 289 et 290, ayant pour résultat un codon d'arrêt au niveau du codon 292 du fait d'un décalage du cadre de lecture,
    (g) transition de C en G au niveau du codon 1338 ayant pour résultat un changement de Glu (CAG) en codon d'arrêt (TAG),
    (h) perte d'un T autour du codon 456 entraínant un décalage du cadre de lecture ayant pour résultat un codon d'arrêt en aval,
    (i) perte de AG autour du codon 243 ayant pour résultat une jonction d'épissure,
    (j) transition de T en C au niveau du codon 437 ayant pour résultat un site donneur d'épissure (CAA/gtaa→CAA/gcaa).
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CA2100472A1 (fr) 1992-07-17
GR3036047T3 (en) 2001-09-28
WO1992013103A1 (fr) 1992-08-06
DK0569527T3 (da) 2001-07-16
DE69231734T2 (de) 2001-09-20
CA2100472C (fr) 2009-04-07
AU1366992A (en) 1992-08-27
EP0569527A1 (fr) 1993-11-18
DE69231734D1 (de) 2001-04-19
ATE199746T1 (de) 2001-03-15
ES2158847T3 (es) 2001-09-16

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